Serine proteases are a class of proteins that proteolytically cleave other proteins. Members of this class of proteins contribute to important biological processes including the proteolytic cascade reactions of complement activation and blood coagulation. Cleavage of a blood coagulation factor contributes to the coagulation cascade, resulting in blood coagulation. A variety of medical conditions can arise where it is advantageous to inhibit the coagulation cascade at the level of one or another proteolytic step. In addition, procedures involving blood product manipulation can activate members of the cascade, and therefore their specific inhibition is advantageous. The neuroprotective effects of serine-proteases have not been so far recognized.
Protein C (PC) is a member of the class of vitamin K-dependent serine protease coagulation factors. Unlike the majority of coagulation factors, such as Factors VIIa, IXa, Xa, XIIa, thrombin, plasmin or plasma kallikrein which are procoagulants, Protein C regulates blood coagulation by acting as a natural anticoagulant that circulates in the blood in an inactive form that requires proteolytic activation to generate the anticoagulant enzyme. The activated form of Protein C, APC, inhibits blood coagulation at the levels of Factors V and VIII in the clotting cascade.
Similar to most other zymogens of extracellular proteases and the above recited blood coagulation factors, Protein C has the core structure of the chymotrypsin family, having insertions and N-terminus extensions that enable regulation of the zymogen and the enzyme (See Owen W., in Hemostasis and Thrombosis: Basic Principles and Clinical Practice, Colman et al., eds, pp. 235–241, J. B. Lippincott Co. (Philadelphia), 1987).
Protein C is composed of domains with discrete structure and function (See Foster et al., Proc. Natl. Acad. Sci. USA. 82:4673–4677 (1985) and Plutzky et al., Proc. Natl. Acad. Sci. USA, 83:546–550 (1986)). The light chain contains an amino-terminal gamma-carboxyglutamic acid (Gla) region, which is followed by two domains that are homologous to domains in the epidermal growth factor (EGF) precursor. The serine protease activity resides in the heavy chain.
The zymogen is activated by the action of thrombin at the site between the arginine residue at position number 15 of the heavy chain and the leucine residue at position 16 (chymotrypsin numbering) (See Kisiel, J. Clin. Invest., 64:761–769, (1976); Marlar et al., Blood, 59:1067–1072 (1982); Fisher et al. Protein Science, 3:588–599 (1994)). Other proteins including Factor Xa (Haley et al., J. Biol. Chem., 264:16303–16310 (1989), Russell's viper venom and trypsin (Esmon et al., J. Biol. Chem., 251:2770–2776 (1976) have also been shown to enzymatically cleave and convert inactive protein C to its activated form. Activated protein C (APC) hydrolyzes arginine esters and related substrates via a core triad of catalytic amino acid residues that occur at Ser-195, His-57, and Asp-102 of the heavy chain (chymotrypsin numbering). The enzyme's specificity is restricted to a small number of protein substrates; blood coagulation cofactors, activated Factors V and VIII, as well as Factors V and VIII are the known macromolecular substrates for the proteolytic inactivation by activated protein C (See Kisiel et al., Biochem., 16:5824–5831 (1977); Vehar et al., Biochem., 19:401–410 (1980); and Walker et al., Biochim. Biophys. Acta., 571:333–342 (1979)).
Thrombin, thought to be the major physiological protein C activator, activates protein C slowly in purified systems, plasma, or blood, when in the presence of physiological concentrations of calcium. A membrane-bound thrombin receptor called thrombomodulin has been identified which accelerates protein C activation. Thrombin binds to thrombomodulin on the luminal surface of endothelial cells and undergoes an increase in specificity for protein C. Calcium is required for this process. Additional studies have revealed that the membrane-lipid binding domain of protein C, the vitamin-K dependent Gla domain, is also required for normal activation of protein C (Esmon et al., in “Progress in Vascular Biology, Hemostasis, and Thrombosis”, Ruggeri et al., eds., Annals of The New York Academy of Sciences, Vol. 614:30–43 (1991)). Endothelial protein C receptor (EPCR) enhances protein C activation by thrombin bound to thrombomodulin.
Thrombosis and thromboembolism, the occurrence of occlusive thrombi in the vasculature of human patients, poses a significant clinical problem and is a significant cause of morbidity and mortality. Arterial thrombi are responsible for myocardial infarction (MI) and cerebral ischemia (stroke), while venous thrombi cause deep vein thrombosis (DVT) and pulmonary embolism (PE). The magnitude of the clinical challenge created by thrombi is reflected in morbidity and mortality statistics. One of the leading causes of death in men over the age of 50 is acute MI, and stroke remains a debilitating and unpredictable disease.
Deep vein thrombosis is a common disease. Well established risk factors include recent surgery, malignant disorders, pregnancy and labor, long term immobilization, and deficiency of one of the main inhibitors of the clotting system. The main inhibitors are known to be protein C, protein S and antithrombin. The causes of deep vein thrombosis in many patients remain unclear. It has recently been established however that a poor anticoagulant response to activated protein C (APC) is present in many families with a hereditary tendency to venous thrombosis.
Inflammation is the body's reaction to injury and infection. Three major events are involved in inflammation: (1) increased blood supply to the injured or infected area; (2) increased capillary permeability enabled by retraction of endothelial cells; and (3) migration of leukocytes out of the capillaries and into the surrounding tissue (hereinafter referred to as cellular infiltration) (Roitt et al., Immunology, l Grower Medical Publishing, New York, 1989).
Increased capillary permeability allows larger molecules to cross the endothelium that are not ordinarily capable of doing so, thereby allowing mediators of immunity such as leukocytes to reach the injured or infected site. Leukocytes, primarily neutrophil polymorphs (also known as polymorphonuclear leukocytes, neutrophils or PMN) and macrophages, migrate to the injured site by a process known as chemotaxis. At the site of inflammation, tissue damage and complement activation cause the release of chemotactic peptides, such as C5a. Complement activation products are also responsible for causing degranulation of phagocytic cells, mast cells and basophils, smooth muscle contraction and increases in vascular permeability (Mulligan et al. J. Immunol. 148:1479–1485 (1991)).
Although leukocyte traversal of vessel walls to extravascular tissue is necessary for host defense against foreign antigens and organisms, leukocyte-endothelial interactions often have deleterious consequences for the host. For example, during the process of adherence and transendothelial migration, leukocytes release oxidants, proteases and cytokines that directly damage endothelium or cause endothelial dysfunction. Once at the extravascular site, emigrated leukocytes further contribute to tissue damage by releasing a variety of inflammatory mediators. Moreover, single leukocytes sticking within the capillary lumen or aggregation of leukocytes within larger vessels are responsible for microvascular occlusion and ischemia. Leukocyte-mediated vascular and tissue injury has been implicated in pathogenesis of a wide variety of clinical disorders, such as acute and chronic allograft rejection, vasculitis, rheumatoid and other forms of inflammatory based arthritis, inflammatory skin diseases, adult respiratory distress syndrome, ischemia-reperfusion syndromes such as myocardial infarction, shock, stroke, organ transplantation, crush injury and limb replantation.
Many other serious clinical conditions involve underlying inflammatory processes in humans. For example, multiple sclerosis (MS) is an inflammatory disease of the central nervous system. In MS, circulating leukocytes infiltrate inflamed brain endothelium and damage myelin, with resultant impaired nerve conduction and paralysis (Yednock et al., Nature 366:63–66 (1992)). Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of tissue damage caused by self antigen directed antibodies. Auto-antibodies bound to antigens in various organs lead to complement-mediated and inflammatory cell mediated tissue damage (Theofilopoubs, A. N., Encyclopedia of Immunology, pp. 1414–1417 (1992)).
Reperfusion injury is another condition associated with activation of the inflammatory system and enhanced leukocyte-endothelial cell (EC) adhesion. There is much evidence that adhesion-promoting molecules facilitate interactions between leukocytes and endothelial cells and play important roles in acute inflammatory reaction and accompanying tissue injury. For example, in acute lung injury caused by deposition of IgG immune complexes or after bolus i.v. infusion of cobra venom factor (CVF), neutrophil activation and the generation of toxic oxygen metabolites cause acute injury (Mulligan et al., J. Immunol. 150(6):2401–2405 (1992)). Neutrophils (PMNs) are also known to mediate ischemia/reperfusion injury in skeletal and cardiac muscle, kidney and other tissues (Pemberton et al., J. Immunol. 150:5104–5113 (1993)).
Infiltration of airways by inflammatory cells, particularly eosinophils, neutrophils and T lymphocytes, is a characteristic feature of atopic or allergic asthma (Cotran et al., Pathological Basis of Disease, W. B. Saunders, Philadelphia, 1994). Cellular infiltration of the pancreas with resultant destruction of islet beta-cells is the underlying pathogenesis associated with insulin-dependent diabetes melitis (Burkly et al., Diabetes 43: 529–534 (1994)). Activation of inflammatory cells whose products cause tissue injury underlies the pathology of inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis (Cotran et al., 1994). Neutrophils, eosinophils, mast cells, lymphocytes and macrophages contribute to the inflammatory response. Minute microabcesses of neutrophils in the upper epithelial layers of the dermis accompany the characteristic epidermal hyperplasia/thickening and scaling in psoriasis.
Various anti-inflammatory drugs are currently available for use in treating conditions involving underlying inflammatory processes. Their effectiveness however, is widely variable and there remains a significant clinical unmet need. This is especially true in the aforementioned diseases where available therapy is either of limited effectiveness or is accompanied by unwanted side effect profiles. Moreover, few clinical agents are available which directly inhibit cellular infiltration, a major underlying cause of tissue damage associated with inflammation. Thus, there is a need for a safe, effective clinical agent for preventing and ameliorating cellular infiltration and consequential pathologic conditions associated with inflammatory diseases, injuries and resultant perturbations of cytokine networks.
Therefore, there is a need in the art for new and better compounds and methods of their use in treating diseases associated with inflammation, thrombosis, and a variety of types of neurological damage.